Chlorin-Vitamin Conjugates

ABSTRACT

The present disclosure relates to novel chlorin-vitamin conjugates and method of making and using the chlorin-vitamin conjugates.

CROSS REFERENCE

The present U.S. patent application is related to and claims thepriority benefit of U.S. Provisional Patent Application Ser. No.62/620,520, filed Jan. 23, 2018, the contents of which is herebyincorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to novel chlorin-vitamin conjugates andmethod of making and using the chlorin-vitamin conjugates.

BACKGROUND

This section introduces aspects that may help facilitate a betterunderstanding of the disclosure. Accordingly, these statements are to beread in this light and are not to be understood as admissions about whatis or is not prior art.

Photodynamic therapy (PDT) is a cancer treatment that involves deliveryof a fluorophore known as photo sensitizer (PS) to tumor tissues uponsystemic administration and activation with visible light (600-800 nm)in the presence of endogenous oxygen. Excitation of PS with light in thered or near infrared (NIR) region generates cytotoxic reactive oxygenspecies (ROS), including singlet oxygen (¹O₂) to cause irreversibledestruction of tumor cells, and to induce immune inflammatory responsesand damage to tumor microvasculature. PDT is an FDA-approved treatmentfor bronchial, esophageal, gastric, cervical, skin, head, and neckcancers. However, PDT has certain limitations. Photosensitizerselectivity and efficacy which continue to be the major barrier forwidespread acceptance of PDT in clinical practice must be improved tooptimize anticancer treatment. Characteristics of ideal photosensitizers include low dark toxicity, high selectivity for tumors overnormal tissues, high quantum yield of light-induced triplet state oxygenformation, and rapid clearance from the body. In PDT and in conventionalcancer chemotherapy, increasing the amount of anticancer drugs is oftennecessary to achieve a linear response in killing cancer cells causingundesirable increase in toxicity to normal healthy cells due tonon-selective tissue targeting.

Targeted therapy for cancer treatment exploiting ligands for selectivedelivery of pharmaceuticals to malignant cells is a strategy to improveanticancer drugs for treatment and imaging. Cancer cells overexpresstumor-specific receptors which can serve as targets to deliver photosensitizers into tumors. In particular, rapidly proliferating cancercells require certain vitamins to sustain growth-causing vitaminreceptors to be overexpressed on the cancer cell surface. B vitaminssuch as folic acid (B9), biotin (B7), riboflavin (B2), and cobalamin(B12) are essential vitamins for survival of all living cells, includingthe growth of cancerous cells. In contrast to folate receptors, whichgained considerable attention as excellent biomarkers for targeted drugdelivery, there are only few studies done on other B vitamin receptors.

There is still a need to develop novel photo sensitizers for PDT thatmay provide low dark toxicity and high selectivity for tumors overnormal tissues.

SUMMARY

One of the primary objectives of the present disclosure is to developnovel photo sensitizers for PDT that may provide low dark toxicity andhigh selectivity for tumors over normal tissues.

In one embodiment, the present disclosure provides a chlorin-vitaminconjugated compound of formula I,

or a pharmaceutically acceptable salt, hydrate, metal complex thereof,

-   wherein-   R¹ is a chlorin moiety;-   R² is a vitamin moiety;-   R³ and R⁴ are independently H or C₁-C₄ branched or straight alkyl;-   L is a branched or straight C₂-C₁₂ linker, wherein at least one    carbon of L is optionally substituted by an oxygen; and-   n is 1-3,-   wherein said chlorin is selected from the group consisting of:

wherein said vitamin is selected from the group consisting of biotin,bexarotene, lipoic acid, pantothenic acid, desthiobiotin, and biocytin;and

-   wherein said chlorin-vitamin conjugated compound is formed through    at least one carboxylic acid group of said chlorin and one    carboxylic acid group of said vitamin.

In one embodiment, the present disclosure provides a method of treatingbreast cancer comprising administering a pharmaceutical composition ofthe chlorin-vitamin conjugated compound of formula I.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodiments, andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of thisdisclosure is thereby intended.

In the present disclosure the term “about” can allow for a degree ofvariability in a value or range, for example, within 10%, within 5%, orwithin 1% of a stated value or of a stated limit of a range.

In the present disclosure the term “substantially” can allow for adegree of variability in a value or range, for example, within 90%,within 95%, or within 99% of a stated value or of a stated limit of arange.

In one embodiment, the present disclosure provides a chlorin-vitaminconjugated compound of formula I,

or a pharmaceutically acceptable salt, hydrate, metal complex thereof,

-   wherein-   R¹ is a chlorin moiety;-   R² is a vitamin moiety;-   R³ and R⁴ are independently H or C₁-C₄ branched or straight alkyl;-   L is a branched or straight C₂-C₁₂ linker, wherein at least one    carbon of L is optionally substituted by an oxygen; and-   n is 1-3,-   wherein said chlorin is selected from the group consisting of:

wherein said vitamin is selected from the group consisting of biotin,bexarotene, lipoic acid, pantothenic acid, desthiobiotin, and biocytin;and

-   wherein said chlorin-vitamin conjugated compound is formed through    at least one carboxylic acid group of said chlorin and one    carboxylic acid group of said vitamin.

In one preferred embodiment, the chlorin is:

In one preferred embodiment, the linker L of formula I is a straight C₆linker.

In one preferred embodiment, when a compound of formula I is a metalcomplex formed by a chlorin moiety and a metal ion, the metal isselected from the group consisting of zinc (Zn), indium (In), palladium(Pd) and platinum (Pt).

In one preferred embodiment, the vitamin is biotin or bexarotene.

In one preferred embodiment, the vitamin is bexarotene.

In one embodiment, the chlorin-vitamin conjugated compound is selectedfrom a group consisting of:

and a pharmaceutically acceptable salt or hydrate thereof.

In one embodiment, the present disclosure provides a method of treatinga disease responsive to the chlorin-vitamin conjugated compound offormula I. In one aspect, the disease is a cancer, wherein the cancermay be but is not limited to head and neck cancer, breast cancer,prostate cancer, lung cancer, liver cancer, gynecological cancer,cervical cancer, brain cancer, melanoma, colorectal cancer bladdercancer, ovarian cancer, or gastrointestinal cancer. In one aspect, thecancer is breast cancer. Examples of breast cancer include, but are notlimited to triple-negative breast cancer (TNBC) or triple-positivebreast cancer TPBC). In one aspect, the breast cancer is triple-negativebreast cancer (TNBC).

In one embodiment, the present disclosure provides that thechlorin-vitamin conjugated compound of claim 1 is used as a photosensitizers for photodynamic therapy (PDT).

In one embodiment, the present disclosure provides that the use of thechlorin-vitamin conjugate composition of the present disclosure in themanufacture of a medicament for the treatment of any cancer as disclosedin the disclosure.

The present disclosure provides pharmaceutical compositions comprising achlorin-vitamin conjugate composition of the present disclosure, and oneor more pharmaceutically acceptable carriers, diluents or excipients.Further, the present disclosure provides a method of treating a canceras disclosed comprising administering to a patient in need thereof apharmaceutical composition of the present disclosure.

Chemistry

Bexarotene was purchased from ChemLeader Biomedical Co. Ltd. (Shanghai,China). All solvents and reagents were purchased mainly from SigmaAldrich Chemical Co. (St. Louis Mo., USA). All air and moisturesensitive reactions were performed in anhydrous solvents under nitrogenatmosphere. Chromatographic purifications were performed in normal phasepreparative TLC (thin-layer chromatography) plate (Analtech). Reactionswere monitored using polyester backed normal phase analytical TLC plate(Merck, Silica gel 60 F₂₅₄ precoated 200 □m) and detected with UV light(□=254 nm). NMR spectra were acquired with a Bruker Avance NMRspectrometer (400 MHz for ¹H, 100 MHz for ¹³C). Chemical shifts arereported in □□ppm referenced according to the deuterated solvents usedas internal standards: CDCl₃ 7.24 ppm (¹H), 77.23 ppm (¹³C). Highresolution mass spectra were obtained on a Bruker micrOTOF-II ESI massspectrometer. All compounds synthesized were isolated and purified in≥95% purity as confirmed by ¹H, ¹³C, 2D COSY (Correlated Spectroscopy),DEPT 135 (Distortion-less Enhancement by Polarization Transfer), HSQC(Heteronuclear Single Quantum Correlation) NMR spectra. Sample puritywas also checked using ThermoScientific Ultimate 3000 HPLC (highperformance liquid chromatography) equipped with a diode-arrayfour-channel variable UV-visible detector, an autosampler and a fractioncollector using a reverse-phase column (C-18, 4.6×50 mm, 3.5 □m) inisocratic mobile phase (100% acetonitrile) visualizing at □=405 and 665nm with a flow rate of 1 mL/min.

Preparation 1: 13¹-Hexamethylenediaminylchlorine₆ dimethyl ester

Methyl pheophorbide a (100 mg, 0.165 mmol) was dissolved in dry CH₂Cl₂and stirred under nitrogen for 10 min. ThenN-t-butyloxycarbonyl-1,6-hexanediamine or (200 mg, 0.93 mmol) dissolvedin 1 mL CHCl₃ (dried over molecular sieves) was added in to the solutionand the mixture was stirred for 24 h. The reaction was monitored by TLC(10% acetone/CH₂Cl₂) until reaction showed disappearance of the startingmaterial methyl pheophorbide a. Additional amine may be added tocomplete the reaction. Solvent was evaporated and the residue waspurified by preparative TLC plate using 8% acetone/CH₂Cl₂ to afford 130mg (0.158 mmol, 96% yield) of 13¹-hexamethylenediaminyl(Boc) chlorine6dimethyl ester. The 13¹-hexamethylenediaminyl (Boc) chlorine6 dimethylester (65 mg, 0.085 mmol) was dissolved in dry CH₂Cl₂ (4 mL) undernitrogen. Reaction mixture was cooled in an ice bath, and TFA (1.2 mL)was added. Nitrogen was removed and the solution was stirred for 3 h.Complete deprotection of Boc group was monitored by TLC (10% acetone inCH₂Cl₂), and then the solution was washed twice with saturated aqueousNaHCO₃ solution. After drying over anhydrous Na₂SO₄, solvent was removedto yield 50 mg (0.075 mmol, 88% yield) of Preparation 1. HRMS(MALDI-TOF) m/z 723.4214 [M]⁺; calcd for C₄₂H₅₄N₆O₅ 723.4228.

EXAMPLE 1: 13¹-Hexamethylenediaminyl-biotinylchlorine₆ dimethyl ester

In a dry round bottom flask containing Preparation 1 (92 mg, 0.128mmol), biotin (48 mg, 0.138 mmol),4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM, 42 mg, 0.150 mmol) was stirred in dry CH₂Cl₂ (25 mL) undernitrogen overnight for 12 h. The reaction was monitored by TLC (10%methanol in CH₂Cl₂) until reaction showed disappearance of the startingamine. Solvent was evaporated and the residue was purified bypreparative TLC plate using the same solvent system to afford 76 mg(0.072 mmol, 57% yield) of Example 1. HRMS (MALDI-TOF) m/z 949.6150[M⁺], calcd for C₅₂H₆₈N₈O₇S 949.2311.

EXAMPLE 2: 13¹-Hexamethylenediaminyl-bexarotenylchlorine₆ dimethylester.

Example 2 was prepared with similar method of preparing Example 1. HRMS(ESI) m/z 1053.6256 [M]⁺, calcd for C₆₆H₈₀N₆O₆ 1053.6212. EXAMPLE 3:Zn(II)-13¹-Hexamethylenediaminyl-Biotinylchlorine₆ dimethyl ester

In a dry round bottom flask, Example 1 (50 mg, 0.053 mmol) was dissolvedin 5 mL methanol. Saturated methanolic solution of zinc(II) acetate (5mL) were added to the mixture and stirred for 2 h. After checkingspectrophotometrically for completion, the reaction mixture was washedwith saturated aqueous solution of sodium bicarbonate, followed bywashing with water (3×50 mL), and extracted with dichloromethane. Theorganic layer was dried over anhydrous sodium sulfate and the organicsolvent was evaporated under high vacuum. The crude product was purifiedby preparative TLC plate using 3% methanol-dichloromethane to afford 40mg (0.039 mmol, 73% yield) of the title compound. HRMS (MALDI-TOF) m/z1033.3986 [M⁺+Na], calcd for C₅₂H₆₆N₈NaO₇SZn 1033.3959.

EXAMPLE 4: Zn(II)-13¹-Hexamethylenediaminyl-bexarotenylchlorine₆dimethyl ester

Example 4 was prepared from Example 2 with similar method of preparingExample 3. HRMS (ESI) m/z 1115.5365 [M⁺+H], calcd for C₆₆H₇₉N₆O₆Zn1115.5347.

EXAMPLE 5: 13¹-Hexamethylenediaminyl-Biotinylchlorine₆ dimethyl esterindium (III) chloride

In a dry round bottom flask, Example 1 (50 mg, 0.053 mmol) was dissolvedin 15 mL toluene, then sodium acetate (500 mg), anhydrous potassiumcarbonate (500 mg) and indium chloride (300 mg) were added. The reactionmixture was refluxed under nitrogen atmosphere overnight. After checkingspectrophotometrically for complete metal insertion, the reactionmixture was neutralized with acetic acid and washed with water (3×50mL), then the organic layer was dried over anhydrous sodium sulfate.Solvent was evaporated under high vacuum. The crude product was purifiedby preparative TLC plate using 3% methanol-dichloromethane to afford 45mg (0.041 mmol, 77% yield) of the title compound.

EXAMPLE 6: 13¹-Hexamethylenediaminyl-bexarotenylchlorine₆ dimethyl esterindium (III) chloride

In a dry round bottom flask, Example 2 (20 mg, 0.019 mmol), sodiumacetate (170 mg), and indium chloride (100 mg) were added, and dissolvedin acetic acid (3 mL). The reaction mixture was refluxed under nitrogenatmosphere overnight. After checking spectrophotometrically for completemetal insertion, the reaction mixture was neutralized with saturatedNaHCO3, and extracted with dichloromethane (20 mL). The organic layerwas washed with water, then dried over anhydrous sodium sulfate. Solventwas evaporated under high vacuum. The crude product was purified bypreparative TLC plate using 4% methanol-dichloromethane to afford 13 mg(0.011 mmol, 57% yield) of the title compound. HRMS (ESI) m/z 1165.5026[M⁺-Cl], calcd for C₆₆H₇₈ClInN₆O₆ 1201.4783.

Assays

In Vitro Cytotoxicity Assay

Mouse colon carcinoma cell line CT26. WT was purchased from the AmericanType Culture Collection (ATCC CRL-2638). CT26 cells were cultured inRPMI 1640 medium (ATCC) and were grown to 80%-90% confluence in 75-cm²culture flasks (Corning, Corning, N.Y., USA) for about a week (5-6 days)in a humidified incubator (Fisher Scientific Isotemp, Waltham, Mass.,USA) with 5% CO₂ at 37° C. During the incubation period, growth mediawas changed once with fresh pre-warmed medium (pH 7.2). To harvest thecells, old growth media was aspirated and 5 mL 0.25% trypsin solution(Thermo Sci Hyclone, Waltham, Mass., USA) were added. The cells wereincubated for 10 min and gently scraped to detach cells from the flaskwalls. Clumped cells were broken up gently and 1 mL of the trypsinizedcell homogenate suspension was transferred into a new T75 cell cultureflask containing pre-warmed media (20 mL) for further culturing.

Human mammary epithelial carcinoma cell lines purchased from theAmerican Type Culture Collection ZR-75-1 (ATCC CRL-1500) and BT-549(ATCC HTB-122) were cultured according to ATCC protocol. Briefly,ZR-75-1 cells were grown in RPMI 1640 medium (ATCC), while BT-549 weregrown in RPMI 1640 containing 0.023 IU/mL insulin. Both weresupplemented with 10% fetal bovine serum (FBS). Cells were grown to80-90% confluence in 75-cm² culture flasks (Corning) for 4-5 days in ahumidified incubator (Fisher Scientific Isotemp) with 5% CO₂ at 37° C.During the incubation period, growth media was changed once with freshpre-warmed media (pH 7.2). To harvest the cells, old growth media wasaspirated out and 3 mL 0.25% trypsin solution (Thermo Sci Hyclone) wereadded. The cells were incubated for 5 min and the cell pellet aftercentrifugation was resuspended in 3 mL media, broken up gently, then 1ml of suspended cells was transferred into a new T75 cell culture flaskcontaining pre-warmed media (20 mL) for further culturing.

Cell Survival Assay

Cells were grown to confluence in a 96-well plate (5×10⁴ cells/well) andtreated for 24 h with compounds or photo sensitizers of varyingconcentrations ranging from 5 nM to 50 μM) in growth media from a stocksolution of 10 mM in DMSO (dimethyl sulfoxide, Fisher). After 24 htreatment, old growth media containing the PS or compounds wereaspirated out and replaced with fresh media. Plates were then positionedbelow a non-coherent LumaCare LC-122 650 nm light source for 1, 2, and 5min at an energy fluence rate of 16 mW/cm² (measured using a Newportoptical power meter Model 840). Unirradiated cells served as controlsamples. The following day, cells were washed with pre-warmed PBS, andMTT (3-[4,5-dimethyl-thiazol-2-yl]-2.5-diphenyltetrazolium bromide,Sigma, 0.3 mg/mL) in PBS was added to each well. Samples were allowed toincubate for additional 2 h, after which dark blue crystals formed. DMSOwas added to each well and plates were shaken at room temperature for 1h to dissolve the purplish-blue formazan crystals. Absorbance values at490 nm were measured on a microplate reader (BioRad 550). Cell survivalwas calculated based on the absorbance of the untreated cells alone (ascontrol) and were directly proportional to the number of viable cells inculture. Results were reported as average of triplicate measurements.

Results from cell viability assay indicated that biotin is not toxic tothe colon cancer cells in the dark and in the presence of light forconcentration of up to 50 μM, which is the maximum concentration usedfor the assay. Compared to the starting methyl pheophorbide (MePheo)which serves as the control, Example 1 increased cell inhibition uponirradiation by approximately 19%. The indium complex, Example 5,compared to either the starting MePheo, the unmetallated Example 1, orthe zinc complex ZnCBTN, Example 3, caused a marked decrease in cellproliferation of colon cancer cells by 30% and 40%-50% upon irradiationfor 2 and 5 min, respectively.

The dark toxicity of the synthesized photo sensitizer linked to biotin(Example 1) or bexarotene (Example 2) compared to the starting methylpheophorbide a (MePheo) after 24-hr treatment of triple-negative breastcancer cells were evaluated. Example 1 has a dose response in theabsence of light when TNBC cells were treated with as low as 5 μMshowing an 86% cell survival, while no significant dark response wasobserved upon treatment with Example 2, Example 4 and MePheo. Example 3,Example 5 and Example 6 also exhibited dark toxicity. The dark toxicityof the synthesized photosensitizers to TNBC was ranked as follows:Example 3 (most phototoxic in the dark)>Example 1>Example 6>Example 5.

For TPBC cells, Example 3 is the most phototoxic in the dark compared tothe other photosensitizers, and can be ranked according to thefollowing: Example 3>Example 5>MePheo>Example 6, and the rest (Example1, Example 2 and Example 4) showing no toxicity in unirradiated TPBCcells. No dark toxicity was observed for all the photosensitizers testedat ≤0.5 μM concentration in TNBC and TPBC cells.

Phototoxicity of Triple-Negative Breast Cancer Cell Line at NanomolarConcentrations. At a low concentration of 100 nM, only Example 1 showeda light-activation dose response when compared to Example 2 and MePheoin TNBC cells with a 60% cell inhibition at 5 min light dose (4.8 Jcm⁻²). However, doubling the concentration to 200 nM, Example 1 is stillmore effective than both Example 2 and MePheo in controlling TNBC cellgrowth with almost 50% reduction in cell proliferation at the lowestlight dose used. All the other photosensitizers were ineffective forTNBC at this nanomolar concentration range. TPBC were totally unaffectedby the other PSs at this low concentrations. Results indicated thatExample 1 is a better photo sensitizer than any of the PSs utilized inthis investigation to inhibit the proliferation of the TNBC cell line.

Fluorescence Microscopy

Fluorescence microscopy was used to visualize and examine the cellmorphological features before and after photosensitization. Specificcellular characteristics can identify the mechanism of cell death whichcan be exploited to understand therapeutic outcome.

Cells (1 mL aliquots) obtained from a diluted cell suspension wereseeded into each well (1.7 cm², 5×10³ cells/well) of a 4-well cultureslide (BD Biosciences) and grown to confluence in 5% CO₂ at 37° C. for3-4 days for attachment to the substratum. After aspirating the oldgrowth media, 1 mL of the compound or photo sensitizer (with varyingconcentrations) in fresh pre-warmed media at 37° C. was added to eachwell. After compound treatment for 24 h, cells were washed twice with 1mL fresh growth media, and then irradiated with light using LumaCareLC-122 as described above. Cells were stained in the dark with Hoechst33258 (Molecular Probes) in pre-warmed media for 10 min at 37° C.,washed twice with filtered PBS, then fixed with filteredparaformaldehyde for 15 min in the incubator. After thorough liquidaspiration, the wells were removed and allowed to air dry in the darkfor 1 h. Slides were protected with coverslips, whose edges were sealedusing a clear fast-drying nail polish and allowed to dry at roomtemperature in the dark for 30 min. Images were recorded usingfluorescence microscopy (DAPI for Hoechst 350-390 nm excitation and460-490 nm emission filters) using an upright fluorescence microscopewith Retiga imaging 2000R (Nikon Optiphot-2, 20× and 40×) and an imageprocessing Nikon NIS-Elements V4.0 Qimaging software.

TABLE 1 Summary of cell survival (%) assay and cell morphologicalfeatures based on fluorescence microscopy images after 24 h compoundtreatment followed by 2 min light irradiation (1.92 J cm⁻²⁾ ontriple-negative BT549 and triple-positive ZR-75-1 breast cancer celllines. Triple-negative breast Triple-positive breast cancer cell cancercell BT549 ZR-75-1 % Cell % Cell Compounds Survival MorphologicalFeatures Survival Morphological Features Example 1 8.1 Chromatin 4.2Chromatin condensation, condensation, nuclear nuclear fragmentationfragmentation Example 3 16 Cell and nuclear 15.4 Cell and nuclearshrinkage, shrinkage, reduced cell reduced cell density density Example5 56 Chromatin 12.9 Cell debris apparent condensation, nuclearfragmentation Example 2 7.7 Chromatin 4.6 Cell and nuclear shrinkage,condensation, nuclear reduced cell density fragmentation Example 4 108Dispersed cells, intact 105 Clustered cells, intact nuclei nuclei,well-defined cytoplasm Example 6 33 Cell and nuclear 80 Clustered cells,intact nuclei shrinkage, reduced cell density Methyl 21 Chromatin 3.4Cell and nuclear shrinkage, pheophorbidea condensation, nuclear reduced(control) fragmentation cell density Bexarotene 111 Dispersed cells,intact 110 Clustered cells, intact (control) nuclei, well-definednuclei, cytoplasm well-defined cytoplasm Biotine 116 Dispersed cells,intact 99 Clustered clumped cells, (control) nuclei, well-defined intactnuclei, cytoplasm well-defined cytoplasm

The novel chlorin-vitamin conjugates disclosed in the present disclosurecan be promising photosensitizers for potential application intriple-negative breast cancer. One preferred compound such as Example 2may be especially useful due to its relatively nonexistent darkcytotoxicity. The chlorin-biotin conjugate Example 1, which exhibited invitro dark cytotoxicity at 5 μM concentration, can be useful for TNBCwith proper adjustment of light dosage since Example 1 showedconsiderable potency at the nanomolar concentration range. Additionally,novel chlorin-vitamin conjugates can have applications fortriple-positive breast cancer (ER⁺/PR⁺/HER2⁺) by targeting the biotinreceptors upregulated in cancer cell surface or the nuclear receptors,respectively, since comparatively no dark toxicity was observed at thehighest concentration of Example 1 and Example 2 used in this study.

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Theimplementations should not be limited to the particular limitationsdescribed. Other implementations may be possible.

1. A chlorin-vitamin conjugated compound of formula I,

or a pharmaceutically acceptable salt, hydrate, metal complex thereof,wherein R¹ is a chlorin moiety; R² is a vitamin moiety; R³ and R⁴ areindependently H or C₁-C₄ branched or straight alkyl; L is a branched orstraight C₂-C₁₂ linker, wherein at least one carbon of L is optionallysubstituted by an oxygen; and n is 1-3, wherein said chlorin is selectedfrom the group consisting of:

wherein said vitamin is selected from the group consisting of biotin,bexarotene, lipoic acid, pantothenic acid, desthiobiotin, and biocytin;and wherein said chlorin-vitamin conjugated compound is formed throughat least one carboxylic acid group of said chlorin and one carboxylicacid group of said vitamin.
 2. The chlorin-vitamin conjugated compoundof claim 1, wherein L is a straight C₆ linker.
 3. The chlorin-vitaminconjugated compound of claim 1, wherein the metal of the metal complexis selected from the group consisting of zinc (Zn), indium (In),palladium (Pd) and platinum (Pt).
 4. The chlorin-vitamin conjugatedcompound of claim 1, wherein the chlorin-vitamin conjugated compound isselected from a group consisting of:

and a pharmaceutically acceptable salt or hydrate thereof.
 5. A methodof treating breast cancer comprising administering a pharmaceuticalcomposition comprising the compound of claim
 1. 6. The method of claim5, wherein the breast cancer is triple-negative breast cancer ortriple-positive breast cancer.
 7. The method of claim 6, wherein thebreast cancer is triple-negative breast cancer.
 8. The method of claim5, wherein the compound of claim 1 is used as a photo sensitizer inphotodynamic therapy (PDT).